Chlorella compositions and methods of use thereof to enhance plant growth

ABSTRACT

The present invention provides a mixture comprising: a) a first liquid composition comprising a culture of microalgae, the microalgae comprising whole pasteurized  Chlorella  cells; and b) a second liquid composition comprising a culture of microalgae, the microalgae comprising lysed pasteurized  Chlorella  cells; wherein a combination of the first liquid composition and the second liquid composition exhibits synergy. Also provided is a method of treating a plant, a plant part, or the locus surrounding the plant to enhance plant growth, the method comprising applying an effective amount of the mixture.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.17/387,970, filed Jul. 28, 2021, which is a continuation-in-part ofInternational Patent Application No. PCT/US2021/036669, filed Jun. 9,2021, which claims benefit of and priority to U.S. Provisional PatentApplication No. 63/148,106, filed Feb. 10, 2021, U.S. Provisional PatentApplication No. 63/056,203, filed Jul. 24, 2020, and U.S. ProvisionalPatent Application No. 63/036,839, filed Jun. 9, 2020. The contents ofeach of the foregoing are incorporated herein by reference in theirentireties.

TECHNICAL FIELD

The present invention relates generally to methods and compositions forstimulating and maintaining enhanced growth in plants. Moreparticularly, the present invention relates to liquid compositionscomprising Chlorella cells that demonstrate plant growth enhancementactivity.

BACKGROUND

It is a common practice in the agricultural field both for foodproduction, ornamental shrubs and trees, and lawn grasses to accelerategrowth by the application of chemical fertilizers, e.g., nitrates,phosphates, and potassium compounds, and chemical materials such aspesticides, herbicides, and fungicides, etc., that can be toxic.Further, it is a present practice to overload the crops with thesechemical materials and to repeatedly treat most crops multiple times ina growing season (typically four times, may be as many as eight timesdepending on the pant and location) because these water-solublesubstances would wash off. The significant amount of runoff means thatusers must use more of these substances and apply more times, whichincreases both the monetary and labor cost. The runoff also results inthese chemical materials finding their way into the soil and the groundwater, and into rivers, lakes, ponds and ultimately the bays and oceans.While these chemicals do enhance the growth of desirable plants, therunoff has toxic effects. Thus, there is a need for environmentallyfriendly and sustainable means for enhancing plant growth.

Chlorella, a genus of single-celled green microalgae, is considered themost photosynthetically efficient organism in the world. Chlorella'schlorophyll content can reach levels as high as 8%; approximately 16times more than most green foods. Chlorella conducts photosynthesisthrough the absorption of sunlight by chlorophyll A, chlorophyll B, andcarotenoid pigments located in its chloroplast.

It has now been recognized that various characteristics including thequality, health, and/or color of plants can be improved through theapplication of effective amounts of biomass that has been obtained fromthe cell tissue of Chlorella species. In addition, application ofChlorella biomass to soil increases soil aggregation and water retentionthereby providing a more productive growth medium for plants. There is aneed to develop effective Chlorella-based agricultural products tosupplement or replace chemical soil amendments and enhance crop growthand yield in a sustainable manner.

SUMMARY

The present invention provides a mixture comprising: a) a first liquidcomposition comprising a culture of microalgae, the microalgaecomprising whole pasteurized Chlorella cells; and b) a second liquidcomposition comprising a culture of microalgae, the microalgaecomprising lysed pasteurized Chlorella cells; wherein a combination ofthe first liquid composition and the second liquid composition exhibitssynergy.

In some aspects, the first liquid composition is formulated forapplication to a plant as a soil drench or in-furrow treatment. In otheraspects, the second liquid composition is formulated for application toa plant propagation material or as a foliar treatment. In one aspect,the second liquid composition is formulated for application as a seedtreatment.

In yet other aspects, the first liquid composition is in a concentrationin the range of 0.003%-0.080% solids by weight. In one aspect, thesecond liquid composition is formulated for application to a plantpropagation material and is in a concentration in the range of 1%-20%solids by weight. In another aspect, the second liquid composition isformulated as a foliar treatment and is in a concentration in the rangeof 0.003%-0.080% solids by weight.

In certain aspects, the whole pasteurized Chlorella cells and/or thelysed pasteurized Chlorella cells are pasteurized at a temperatureranging from 50° C. to 90° C. In other aspects, the lysed pasteurizedChlorella cells are lysed with a bead mill, a shear mill, a pulsedelectron field (PEF), high pressure homogenization, an enzyme, achemical solvent, or a combination thereof.

In one aspect, the ratio by weight of whole pasteurized Chlorella cellsto lysed pasteurized Chlorella cells is from 1:500 to 500:1.

In another aspect, the first liquid composition and/or the second liquidcomposition further comprise at least one culture stabilizer selectedfrom the group consisting of potassium sorbate, phosphoric acid,ascorbic acid, sodium benzoate, and any combination thereof.

In some aspects, the present invention relates to a plant propagationmaterial treated with a mixture disclosed herein in an amount of from0.01 g to 10 kg per 100 kg of plant propagation material. In one aspect,the plant propagation material is a cover crop. In another aspect, thecover crop is clover or cereal rye. In another aspect, the cover crop ispasture.

In other aspects, the present invention relates to a kit for preparingan agricultural composition, the kit comprising: a) a compositioncomprising the first liquid composition as defined herein and at leastone auxiliary; and b) a composition comprising the second liquidcomposition as defined herein and at least one auxiliary.

In one aspect, the present invention provides a mixture comprising: a) afirst liquid composition formulated for application to a plant as a soildrench or in-furrow treatment comprising a culture of microalgae, themicroalgae comprising whole pasteurized Chlorella cells; and b) a secondliquid composition formulated for application as a seed treatmentcomprising a culture of microalgae, the microalgae comprising lysedpasteurized Chlorella cells; wherein a combination of the first liquidcomposition and the second liquid composition exhibits synergy.

In another aspect, the present invention relates to a method of treatinga plant, a plant part, or the locus surrounding the plant to enhanceplant growth or chlorophyll content, the method comprising applying aneffective amount of a mixture comprising: a) a first liquid compositiontreatment comprising a culture of microalgae, the microalgae comprisingwhole pasteurized Chlorella cells; and b) a second liquid compositiontreatment comprising a culture of microalgae, the microalgae comprisinglysed pasteurized Chlorella cells.

In one aspect, the first liquid composition is applied as a soil drenchor in-furrow treatment and the second liquid composition is applied to aplant propagation material or as a foliar treatment.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the viscosity of different preparations of PHYCOTERRA® ST(lysed Chlorella microalgae) between about pH 2 and about pH 7 without awashing step. Each line represents a different preparation.

FIG. 2 depicts a significant decrease in viscosity after a washing stepwith a preparation of PHYCOTERRA® ST (lysed Chlorella microalgae).

FIG. 3 depicts the growth and movement of non-pathogenic microbes on asoft agar plate (0.5% agar) towards a seed coated with PHYCOTERRA® ST(lysed Chlorella microalgae) after 22 hours.

FIG. 4 depicts the culturable bacterial populations obtained aftertreatment of soil samples (i.e., loam soil) from Granger, Iowa withPHYCOTERRA® (whole cell Chlorella microalgae) or PHYCOTERRA® ST (lysedChlorella microalgae) compared to those obtained from the same soil leftuntreated.

FIG. 5 depicts the culturable bacterial populations obtained aftertreatment of soil samples (i.e., sand soil) from Douglas, Georgia withPHYCOTERRA® (whole cell Chlorella microalgae) or PHYCOTERRA® ST (lysedChlorella microalgae) compared to those obtained from the same soil leftuntreated.

FIG. 6 depicts the total bacterial populations and Bacillus sp.populations obtained after treatment of soil samples (i.e., sand soil)from Douglas, Ga. with PHYCOTERRA® (whole cell Chlorella microalgae) orPHYCOTERRA® ST (lysed Chlorella microalgae) compared to those obtainedfrom the same soil left untreated.

FIG. 7 depicts the total bacterial populations and Pseudomonas sp.populations obtained after treatment of soil samples (i.e., loam soil)from Granger, Iowa with PHYCOTERRA® (whole cell Chlorella microalgae) orPHYCOTERRA® ST (lysed Chlorella microalgae) compared to those obtainedfrom the same soil left untreated

FIG. 8 depicts the shoot biomass of corn plants without treatment(“Control”), with an in-furrow treatment of PHYCOTERRA® (whole cellChlorella microalgae), or with a seed treatment of PHYCOTERRA® ST (lysedChlorella microalgae). Differences in letters above each bar indicate astatistical difference in comparison to control (p=0.0979).

FIG. 9 depicts a photograph of representative lettuce plants leftuntreated (“Control”), treated with a drenching application ofPHYCOTERRA® (whole cell Chlorella microalgae), or treated with adrenching application of PHYCOTERRA® ST (lysed Chlorella microalgae).Average increases in shoot biomass compared to control plants are shownbelow the photograph.

FIG. 10 depicts the shoot biomasses of a clover cover crop withouttreatment (“Control”) or with a seed treatment of PHYCOTERRA® ST (lysedChlorella microalgae).

FIG. 11 depicts the shoot biomasses of a cereal rye cover crop withouttreatment (“Control”) or with a seed treatment of PHYCOTERRA® ST (lysedChlorella microalgae).

FIG. 12 depicts photographs of representative wheat plants aftertreatment with a standard fungicide and insecticide alone or incombination with PHYCOTERRA® ST (lysed Chlorella microalgae).

FIG. 13 depicts the average yield in bushels per acre across three fieldtrials in Hassel, N.C.; Kenansville, N.C.; and Burgaw, N.C. with wheattreated with a standard fungicide and insecticide alone or incombination with PHYCOTERRA® ST (lysed Chlorella microalgae).

FIG. 14 depicts photographs of corn plant shoots without treatment(“Control”), with a treatment of PHYCOTERRA® ST (lysed Chlorellamicroalgae) applied to seeds, or with a combination treatment ofPHYCOTERRA® ST (lysed Chlorella microalgae) applied to seeds andPHYCOTERRA® (whole cell Chlorella microalgae) applied as a soil drench.The corn plants shown are at V-9 stage of growth.

FIG. 15 depicts photographs of corn plant roots without treatment(“Control”), with a treatment of PHYCOTERRA® ST (lysed Chlorellamicroalgae) applied to seeds, or with a combination treatment ofPHYCOTERRA® ST (lysed Chlorella microalgae) applied to seeds andPHYCOTERRA® (whole cell Chlorella microalgae) applied as a soil drench.The corn plants shown are at V-9 stage of growth.

DETAILED DESCRIPTION

As used herein and in the appended claims, the singular forms “a,” “an,”and “the” include plural referents unless the context clearly dictatesotherwise. For example, “a” or “an” means “at least one” or “one ormore.”

Throughout this disclosure, various aspects of the claimed subjectmatter are presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theclaimed subject matter. Accordingly, the description of a range shouldbe considered to have specifically disclosed all the possible sub-rangesas well as individual numerical values within that range. For example,where a range of values is provided, it is understood that eachintervening value, between the upper and lower limit of that range andany other stated or intervening value in that stated range isencompassed within the claimed subject matter. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the claimed subjectmatter, subject to any specifically excluded limit in the stated range.Where the stated range includes one or both of the limits, rangesexcluding either or both of those included limits are also included inthe claimed subject matter. This applies regardless of the breadth ofthe range.

The term “microalgae” as used herein refers to microscopic single cellorganisms such as microalgae, cyanobacteria, algae, diatoms,dinoflagellates, freshwater organisms, marine organisms, or othersimilar single cell organisms capable of growth in phototrophic,mixotrophic, or heterotrophic culture conditions.

The term “plant propagation material” is to be understood to denote allthe generative parts of the plant such as seeds and vegetative plantmaterial such as cuttings and tubers (e. g. potatoes), which can be usedfor the multiplication of the plant. This includes seeds, roots, fruits,tubers, bulbs, rhizomes, shoots, sprouts and other parts of plants,including seedlings and young plants, which are to be transplanted aftergermination or after emergence from soil.

The term “auxiliary” as used herein refers to an inert ingredientcommonly used in agricultural compositions. Examples of auxiliariesinclude, but are not limited to, extenders, solvents, diluents,emulsifiers, dispersants, binders, fixing agents, wetting agents, dyes,pigments, antifoams, preservatives, secondary thickeners, and stickers.

Analysis of the DNA sequence of the strain of Chlorella sp. describedherein was done in the NCBI 18s rDNA reference database at the CultureCollection of Algae at the University of Cologne (CCAC) and showedsubstantial similarity (i.e., greater than 95%) with multiple knownstrains of Chlorella and Micractinium. Those of skill in the art willrecognize that Chlorella and Micractinium appear closely related in manytaxonomic classification trees for microalgae, and strains and speciesmay be re-classified from time to time within the Chlorella andMicractinium genera. As would be understood in the art, thereclassification of various taxa is not unusual, and occurs asdevelopments in science are made. Any disclosure in the specificationregarding the classification of exemplary species or strains should beviewed in light of such developments. While the exemplary microalgaestrain is referred to in the instant specification as Chlorella, it isrecognized that microalgae strains in related taxonomic classificationswith similar characteristics to the exemplary microalgae strain wouldreasonably be expected to produce similar results. Accordingly, anymention of Chlorella herein should be understood to include Micractiniumspecies genetically and morphologically similar to species classifiedwithin the genus Chlorella as of the filing date.

By artificially controlling aspects of the microalgae culturing processsuch as the organic carbon feed (e.g., acetic acid, acetate), oxygenlevels, pH, and light, the culturing process differs from the culturingprocess that microalgae experiences in nature. In addition tocontrolling various aspects of the culturing process, intervention byhuman operators or automated systems occurs during the non-axenicmixotrophic culturing of microalgae through contamination controlmethods to prevent the microalgae from being overrun and outcompeted bycontaminating organisms (e.g., fungi, bacteria). Contamination controlmethods for microalgae cultures are known in the art and such suitablecontamination control methods for non-axenic mixotrophic microalgaecultures are disclosed in W02014/074769A2 (Ganuza, et al.), herebyincorporated by reference. By intervening in the microalgae culturingprocess, the impact of the contaminating microorganisms can be mitigatedby suppressing the proliferation of containing organism populations andthe effect on the microalgal cells (e.g., lysing, infection, death,clumping). Thus, through artificial control of aspects of the culturingprocess and intervening in the culturing process with contaminationcontrol methods, the microalgae culture produced as a whole and used inthe described inventive compositions differs from the culture thatresults from a microalgae culturing process that occurs in nature.

In some embodiments and Examples below, the microalgae composition maybe referred to as PHYCOTERRA® or PHYCOTERRA® ST. The PHYCOTERRA® orPHYCOTERRA® ST Chlorella microalgae composition is a microalgaecomposition comprising Chlorella. The PHYCOTERRA® product contains wholecell Chlorella biomass while the PHYCOTERRA® ST contains lysed cellChlorella biomass. The PHYCOTERRA® Chlorella microalgae compositiontreatments were prepared by growing the Chlorella in non-axenic aceticacid supplied mixotrophic conditions, increasing the concentration ofChlorella using a centrifuge, pasteurizing the concentrated Chlorella atbetween 65° C.-75° C. for between 90-150 minutes, adding potassiumsorbate and phosphoric acid to stabilize the pH of the Chlorella, andthen adjusting the whole biomass treatment to the desired concentration.The PHYCOTERRA® Chlorella microalgae composition may compriseapproximately 10% w/w of Chlorella microalgae cells. Furthermore, thePHYCOTERRA® Chlorella microalgae composition may comprise betweenapproximately 0.3% potassium sorbate and between approximately 0.5%-1.5%phosphoric acid to stabilize the pH of the Chlorella to between 3.0-4.0and 88.2%-89.2% water. It should be clearly understood, however, thatother variations of the PHYCOTERRA® Chlorella microalgae composition,including variations in the microalgae strains, variations in thestabilizers, and/or variations in the % composition of each componentmay be used and may achieve similar results.

In some embodiments and Examples below, the microalgae composition maybe an OMRI certified microalgae composition referred to as TERRENE®. TheOMRI certified TERRENE® Chlorella microalgae composition is a microalgaecomposition comprising Chlorella. The OMRI certified TERRENE® Chlorellamicroalgae composition treatments were prepared by growing the Chlorellain non-axenic acetic acid supplied mixotrophic conditions, increasingthe concentration of Chlorella using a centrifuge, pasteurizing theconcentrated Chlorella at between 65° C.-75° C. for between 90-150minutes, adding citric acid to stabilize the pH of the Chlorella, andthen adjusting the whole biomass treatment to the desired concentration.The OMRI certified TERRENE® Chlorella microalgae composition maycomprise approximately 10% w/w of Chlorella microalgae cells.Furthermore, the OMRI certified TERRENE® Chlorella microalgaecomposition may comprise between approximately 0.5%-2.0% citric acid tostabilize the pH of the Chlorella to between 3.0-4.0 and 88%-89.5%water. It should be clearly understood, however, that other variationsof the OMRI certified TERRENE® Chlorella microalgae composition,including variations in the microalgae strains, variations in thestabilizers, and/or variations in the % composition of each componentmay be used and may achieve similar results.

In some embodiments and Examples below, the microalgae composition maybe an OMRI certified microalgae composition referred to as OMRIcertified TERRENE® Chlorella pasteurized at 65° C. microalgaecomposition or as TERRENE65. The OMRI certified TERRENE® Chlorellapasteurized at 65° C. microalgae composition is a microalgae compositioncomprising Chlorella. The OMRI certified TERRENE® Chlorella pasteurizedat 65° C. microalgae composition treatments were prepared by growing theChlorella in non-axenic acetic acid supplied mixotrophic conditions,increasing the concentration of Chlorella using a centrifuge,pasteurizing the concentrated Chlorella at 65° C. for between 90-150minutes, adding citric acid to stabilize the pH of the Chlorella, andthen adjusting the whole biomass treatment to the desired concentration.The OMRI certified TERRENE® Chlorella pasteurized at 65° C. microalgaecomposition may comprise approximately 10% w/w of Chlorella microalgaecells. Furthermore, the OMRI certified TERRENE® Chlorella pasteurized at65° C. microalgae composition may comprise between approximately0.5%-2.0% citric acid to stabilize the pH of the Chlorella to between3.0-4.0and 88-89.5% water. It should be clearly understood, however,that other variations of the OMRI certified TERRENE® Chlorellapasteurized at 65° C. microalgae composition, including variations inthe microalgae strains, variations in the stabilizers, variations in thepasteurization temperature, and/or variations in the % composition ofeach component may be used and may achieve similar results.

In some embodiments and Examples below, the microalgae composition maybe an OMRI certified microalgae composition referred to as OMRIcertified TERRENE® Chlorella pasteurized at 90° C. microalgaecomposition or as TERRENE90. The OMRI certified TERRENE® Chlorellapasteurized at 90° C. microalgae composition is a microalgae compositioncomprising Chlorella. The OMRI certified TERRENE® Chlorella pasteurizedat 90° C. microalgae composition treatments were prepared by growing theChlorella in non-axenic acetic acid supplied mixotrophic conditions,increasing the concentration of Chlorella using a centrifuge,pasteurizing the concentrated Chlorella at 90° C. for between 90-150minutes, adding citric acid to stabilize the pH of the Chlorella, andthen adjusting the whole biomass treatment to the desired concentration.The OMRI certified TERRENE® Chlorella pasteurized at 90° C. microalgaecomposition may comprise approximately 10% w/w of Chlorella microalgaecells. Furthermore, the OMRI certified TERRENE® Chlorella pasteurized at90° C. microalgae composition may comprise between approximately0.5%-2.0% citric acid to stabilize the pH of the Chlorella to between3.0-4.0 and 88-89.5% water. It should be clearly understood that othervariations of the OMRI certified TERRENE® Chlorella pasteurized at 90°C. microalgae composition, including variations in the microalgaestrains, variations in the stabilizers, variations in the pasteurizationtemperature, and/or variations in the % composition of each componentmay be used and may achieve similar results.

A liquid composition comprising microalgae can be stabilized by heatingand cooling in a pasteurization process. In certain aspects, the activeingredients of the microalgae-based compositions maintain effectivenessin enhancing at least one characteristic of a plant after beingsubjected to the heating and cooling of a pasteurization process. Inother embodiments, liquid compositions with whole cells or processedcells (e.g., dried, lysed, extracted) of microalgae cells may not needto be stabilized by pasteurization. For example, microalgae cells thathave been processed, such as by drying, lysing, and extraction, orextracts can include such low levels of bacteria that a liquidcomposition can remain stable without being subjected to the heating andcooling of a pasteurization process.

In some embodiments, the composition is lysed. Lysing is a techniquewhere the cell membrane of a cell is ruptured, which releases lysate,the fluid contents of lysed cells, from the cells. As an example, thelysing process can comprise anything suitable that ruptures a cellmembrane. For example, a bead mill may be used for lysing, wherefeedstock biomass solids can be dispersed and wetted (e.g., placed intoa liquid phase). In this example the bead mill can utilize ceramic,glass, or metal beats (e.g., of a suitable size for the desired result)disposed in a chamber, such as a rotating cylinder, to collide with andmechanically macerate the solid biomass in the mill, which can helprupture the cell walls (e.g., the hydrogen bonds that hold together acell membrane). Accordingly, in this example, the whole biomass may belysed with water at cooler temperatures, with the resulting lysatecomprising lipids in the form of an oil, biomass cell contents andunbroken biomass solid (e.g., non-target portion of biomass), and water.

In another aspect, the biomass is lysed using a shear mill. A shear millutilizes a rotating impeller or high-speed rotor to create flow andshear of its contents. This causes the solid particles, such as biomasssolid, to rupture due to shear stress.

In another aspect, the biomass is lysed using a pulsed electron field(PEF), high pressure homogenization, enzymes, and/or a chemical means(e.g., with a solvent).

In some embodiments, the composition can be heated to a temperature inthe range of 50-130° C. In some embodiments, the composition can beheated to a temperature in the range of 55-65° C. In some embodiments,the composition can be heated to a temperature in the range of 58-62° C.In some embodiments, the composition can be heated to a temperature inthe range of 50-60° C. In some embodiments, the composition can beheated to a temperature in the range of 60-90° C. In some embodiments,the composition can be heated to a temperature in the range of 70-80° C.In some embodiments, the composition can be heated to a temperature inthe range of 100-150° C. In some embodiments, the composition can beheated to a temperature in the range of 120-130° C.

In some embodiments, the composition can be heated for a time period inthe range of 1-150 minutes. In some embodiments, the composition can beheated for a time period in the range of 110-130 minutes. In someembodiments, the composition can be heated for a time period in therange of 90-100 minutes. In some embodiments, the composition can beheated for a time period in the range of 100-110 minutes. In someembodiments, the composition can be heated for a time period in therange of 110-120 minutes. In some embodiments, the composition can beheated for a time period in the range of 120-130 minutes. In someembodiments, the composition can be heated for a time period in therange of 130-140 minutes. In some embodiments, the composition can beheated for a time period in the range of 140-150 minutes. In someembodiments, the composition is heated for less than 15 min. In someembodiments, the composition is heated for less than 2 min.

After the step of heating or subjecting the liquid composition to hightemperatures is complete, the compositions can be cooled at any rate toa temperature that is safe to work with. In one non-limiting embodiment,the composition can be cooled to a temperature in the range of 35-45° C.In some embodiments, the composition can be cooled to a temperature inthe range of 36-44° C. In some embodiments, the composition can becooled to a temperature in the range of 37-43° C. In some embodiments,the composition can be cooled to a temperature in the range of 38-42° C.In some embodiments, the composition can be cooled to a temperature inthe range of 39-41° C. In further embodiments, the pasteurizationprocess can be part of a continuous production process that alsoinvolves packaging, and thus the liquid composition can be packaged(e.g., bottled) directly after the heating or high temperature stagewithout a cooling step.

In some embodiments, the composition can include 2.5-30% solids byweight of microalgae cells (i.e., 2.5-30 g of microalgae cells/100 mL ofthe liquid composition). In some embodiments, the composition caninclude 2.5-5% solids by weight of microalgae cells (i.e., 2.5-5 g ofmicroalgae cells/100 mL of the liquid composition). In some embodiments,the composition can include 5-20% solids by weight of microalgae cells.In some embodiments, the composition can include 5-15% solids by weightof microalgae cells. In some embodiments, the composition can include5-10% solids by weight of microalgae cells. In some embodiments, thecomposition can include 10-20% solids by weight of microalgae cells. Insome embodiments, the composition can include 10-20% solids by weight ofmicroalgae cells. In some embodiments, the composition can include20-30% solids by weight of microalgae cells. In some embodiments,further dilution of the microalgae cells percent solids by weight canoccur before application for low concentration applications of thecomposition.

In some embodiments, the composition can include less than 1% by weightof microalgae biomass or extracts (i.e., less than 1 g of microalgaederived product/100 mL of the liquid composition). In some embodiments,the composition can include less than 0.9% by weight of microalgaebiomass or extracts. In some embodiments, the composition can includeless than 0.8% by weight of microalgae biomass or extracts. In someembodiments, the composition can include less than 0.7% by weight ofmicroalgae biomass or extracts. In some embodiments, the composition caninclude less than 0.6% by weight of microalgae biomass or extracts. Insome embodiments, the composition can include less than 0.5% by weightof microalgae biomass or extracts. In some embodiments, the compositioncan include less than 0.4% by weight of microalgae biomass or extracts.In some embodiments, the composition can include less than 0.3% byweight of microalgae biomass or extracts. In some embodiments, thecomposition can include less than 0.2% by weight of microalgae biomassor extracts. In some embodiments, the composition can include less than0.1% by weight of microalgae biomass or extracts. In some embodiments,the composition can include at least 0.0001% by weight of microalgaebiomass or extracts. In some embodiments, the composition can include atleast 0.001% by weight of microalgae biomass or extracts. In someembodiments, the composition can include at least 0.01% by weight ofmicroalgae biomass or extracts. In some embodiments, the composition caninclude at least 0.1% by weight of microalgae biomass or extracts. Insome embodiments, the composition can include 0.0001-1% by weight ofmicroalgae biomass or extracts. In some embodiments, the composition caninclude 0.0001-0.001% by weight of microalgae biomass or extracts. Insome embodiments, the composition can include 0.001-0.01% by weight ofmicroalgae biomass or extracts. In some embodiments, the composition caninclude 0.01-0.1% by weight of microalgae biomass or extracts. In someembodiments, the composition can include 0.1-1% by weight of microalgaebiomass or extracts.

In some embodiments, an application concentration of 0.1% of microalgaebiomass or extract equates to 0.04 g of microalgae biomass or extract in40 mL of a composition. While the desired application concentration to aplant can be 0.1% of microalgae biomass or extract, the composition canbe packaged as a 10% concentration (0.4 mL in 40 mL of a composition).Thus, a desired application concentration of 0.1% would require 6,000 mLof the 10% microalgae biomass or extract in the 100 gallons of waterapplied to the assumption of 15,000 plants in an acre, which isequivalent to an application rate of about 1.585 gallons per acre. Insome embodiments, a desired application concentration of 0.01% ofmicroalgae biomass or extract using a 10% concentration compositionequates to an application rate of about 0.159 gallons per acre. In someembodiments, a desired application concentration of 0.001% of microalgaebiomass or extract using a 10% concentration composition equates to anapplication rate of about 0.016 gallons per acre. In some embodiments, adesired application concentration of 0.0001% of microalgae biomass orextract using a 10% concentration composition equates to an applicationrate of about 0.002 gallons per acre.

In another non-limiting embodiment, correlating the application of themicroalgae biomass or extract on a per plant basis using the assumptionof 15,000 plants per acre, the composition application rate of 1 gallonper acre is equal to about 0.25 mL per plant=0.025 g per plant=25 mg ofmicroalgae biomass or extract per plant. The water requirementassumption of 100 gallons per acre is equal to about 35 mL of water perplant. Therefore, 0.025 g of microalgae biomass or extract in 35 mL ofwater is equal to about 0.071 g of microalgae biomass or extract per 100mL of composition equates to about a 0.07% application concentration. Insome embodiments, the microalgae biomass or extract based compositioncan be applied at a rate in a range as low as about 0.001-10 gallons peracre, or as high as up to 150 gallons per acre.

In some embodiments, the applications are performed using a 10% solidssolution by weight microalgae composition. For greenhouse trials, therates vary and essentially refer to how much volume of the 10% solidssolution are added in a given volume of water (e.g., 2.5% vv-5.0% v/v).

According to one embodiment, individual components of the compositionaccording to the invention such as parts of a kit or parts of a binarymixture may be mixed by the user himself in a spray tank or any otherkind of vessel used for applications (e.g., seed treater drums, seedpelleting machinery, knapsack sprayer) and further auxiliaries may beadded, if appropriate. Consequently, one embodiment of the invention isa kit for preparing an agricultural composition, the kit comprising a) acomposition comprising whole pasteurized Chlorella cells as definedherein and at least one auxiliary; and/or b) a composition comprisinglysed pasteurized Chlorella cells as defined herein and at least oneauxiliary.

Additionally, the present invention is directed to a method of treatinga plant, a plant part, such as a seed, root, rhizome, corm, bulb, ortuber, and/or a locus on which or near which the plant or the plantparts grow, such as soil, to enhance plant growth comprising the step ofsimultaneously or sequentially applying to a plant, a plant part and/ora plant loci: a) a first liquid composition comprising a culture ofmicroalgae, the microalgae comprising whole pasteurized Chlorella cells;and b) a second liquid composition comprising a culture of microalgae,the microalgae comprising lysed pasteurized Chlorella cells.

The compositions disclosed herein may be applied in any desired manner,such as in the form of a seed coating, soil drench, and/or directlyin-furrow and/or as a foliar spray and applied either pre-emergence,post-emergence or both. In other words, the compositions can be appliedto the seed, the plant or to the soil wherein the plant is growing orwherein it is desired to grow (plant's locus of growth).

In some embodiments, the liquid microalgae-based composition may beapplied to soil, seeds, and plants in an in-furrow application. Anapplication of the microalgae-based composition in-furrow requires a lowamount of water and targets the application to a small part of thefield. The application in-furrow also concentrates the application ofthe microalgae-based composition at a place where the seedling radiclesand roots will pick up the material in the composition or make use ofcaptured nutrients, including phytohormones.

In some embodiments, the liquid microalgae-based composition may beapplied to soil, seeds, and plants as a side dress application. One ofthe principals of plant nutrient applications is to concentrate thenutrients in an area close to the root zone so that the plant roots willencounter the nutrients as the plant grows. Side-dress applications usea “knife” that is inserted into the soil and delivers the nutrientsaround 2 inches along the row and about 2 inches or more deep.Side-dress applications are made when the plants are young and prior toflowering to support yield. Side-dress applications can only be madeprior to planting in drilled crops, i.e. wheat and other grains, andalfalfa, but in row crops such as peppers, corn, tomatoes they can bemade after the plants have emerged.

In some embodiments, the liquid microalgae-based composition may beapplied to soil, seeds, and plants through a drip system. Depending onthe soil type, the relative concentrations of sand, silt and clay, andthe root depth, the volume that is irrigated with a drip system may beabout ⅓ of the total soil volume. The soil has an approximate weight of4,000,000 lbs. per acre one foot deep. Because the roots grow wherethere is water, the plant nutrients in the microalgae-based compositionwould be delivered to the root system where the nutrients will impactmost or all of the roots. Experimental testing of different applicationrates to develop a rate curve would aid in determining the optimum rateapplication of a microalgae-based composition in a drip systemapplication.

In some embodiments, the liquid microalgae-based composition may beapplied to soil, seeds, and plants through a pivot irrigationapplication. The quantity and frequency of water delivered over an areaby a pivot irrigation system is dependent on the soil type and crop.Applications may be 0.5 inch or more and the exact demand for water canbe quantitatively measured using soil moisture gauges. For crops such asalfalfa that are drilled in (very narrow row spacing), the roots occupythe entire soil area. Penetration of the soil by the microalgae-basedcomposition may vary with a pivot irrigation application but would beeffective as long as the application can target the root system of theplants. In some embodiments, the microalgae-based composition may beapplied in a broadcast application to plants with a high concentrationof plants and roots, such as row crops.

In certain aspects, the liquid microalgae-based composition is appliedat 0.1-150 gallons per acre, 0.1-50 gallons per acre, or 0.1-10 gallonsper acre.

In certain aspects, the present invention provides a method of treatinga plant, a plant part, or the locus surrounding the plant to enhanceplant growth, the method comprising applying an effective amount of amixture comprising: a) a first liquid composition treatment comprising aculture of microalgae, the microalgae comprising whole pasteurizedChlorella cells; and b) a second liquid composition treatment comprisinga culture of microalgae, the microalgae comprising lysed pasteurizedChlorella cells. In one aspect, the presence of the whole pasteurizedChlorella cells and the lysed pasteurized Chlorella cells in the firstliquid composition and the second liquid composition enhances the growthof the plant compared to compositions comprising non-pasteurizedChlorella cells, which lack the whole pasteurized Chlorella cells andthe lysed pasteurized Chlorella cells. In other aspects, the applicationof the first liquid composition and the second liquid compositionproduces a synergistic enhancement of plant growth.

The present invention involves the use of a microalgae composition.Microalgae compositions, methods of preparing liquid microalgaecompositions, and methods of applying the microalgae compositions toplants are disclosed in WO 2017/218896 A1 (Shinde et al.) entitled“Microalgae-Based Composition, and Methods of its Preparation andApplication to Plants,” which is incorporated herein in full byreference. In one or more embodiments, the microalgae composition maycomprise approximately 10%-10.5% w/w of Chlorella microalgae cells. Inone or more embodiments, the microalgae composition may also compriseone of more stabilizers, such as potassium sorbate, phosphoric acid,ascorbic acid, sodium benzoate, citric acid, or the like, or anycombination thereof. For example, in one or more embodiments, themicroalgae composition may comprise approximately 0.3% w/w of potassiumsorbate or another similar compound to stabilize its pH and may furthercomprise approximately 0.5-1.5% w/w phosphoric acid or another similarcompound to prevent the growth of contaminants. As a further example, inone or more embodiments where it is desired to use an OMRI (OrganicMaterials Review Institute) certified organic composition, themicroalgae composition may comprise 1.0-2.0% w/w citric acid tostabilize its pH and may not contain potassium sorbate or phosphoricacid. In one or more embodiments, the pH of the microalgae compositionmay be stabilized to between 3.0-4.0.

In some embodiments, the composition is a liquid and substantiallyincludes of water.

In some embodiments, the composition can include 70-99% water. In someembodiments, the composition can include 85-95% water. In someembodiments, the composition can include 70-75% water. In someembodiments, the composition can include 75-80% water. In someembodiments, the composition can include 80-85% water. In someembodiments, the composition can include 85-90% water. In someembodiments, the composition can include 90-95% water. In someembodiments, the composition can include 95-99% water. The liquid natureand high-water content of the composition facilitates administration ofthe composition in a variety of manners, such as but not limit to:flowing through an irrigation system, flowing through an above grounddrip irrigation system, flowing through a buried drip irrigation system,flowing through a central pivot irrigation system, sprayers, sprinklers,and water cans.

In some embodiments, the liquid composition can be used immediatelyafter formulation, or can be stored in containers for later use. In someembodiments, the composition can be stored out of direct sunlight. Insome embodiments, the composition can be refrigerated. In someembodiments, the composition can be stored at 1-10° C. In someembodiments, the composition can be stored at 1-3° C. In someembodiments, the composition can be stored at 3-50° C. In someembodiments, the composition can be stored at 5-8° C. In someembodiments, the composition can be stored at 8-10° C.

In some embodiments, administration of the liquid composition to soil, aseed or plant can be in an amount effective to produce an enhancedcharacteristic in plants compared to a substantially identicalpopulation of untreated seeds or plants. Such enhanced characteristicscan include accelerated seed germination, accelerated seedlingemergence, improved seedling emergence, improved leaf formation,accelerated leaf formation, improved plant maturation, accelerated plantmaturation, increased plant yield, increased plant growth, increasedplant quality, increased plant health, increased fruit yield, increasedfruit sweetness, increased fruit growth, and increased fruit quality.Non-limiting examples of such enhanced characteristics can includeaccelerated achievement of the hypocotyl stage, accelerated protrusionof a stem from the soil, accelerated achievement of the cotyledon stage,accelerated leaf formation, increased marketable plant weight, increasedmarketable plant yield, increased marketable fruit weight, increasedproduction plant weight, increased production fruit weight, increasedutilization (indicator of efficiency in the agricultural process basedon ratio of marketable fruit to unmarketable fruit), increasedchlorophyll content (indicator of plant health), increased plant weight(indicator of plant health), increased root weight (indicator of planthealth), increased shoot weight (indicator of plant health), increasedplant height, increased thatch height, increased resistance to saltstress, increased plant resistance to heat stress (temperature stress),increased plant resistance to heavy metal stress, increased plantresistance to drought, increased plant resistance to disease, improvedcolor, reduced insect damage, reduced blossom end rot, and reduced sunburn. Such enhanced characteristics can occur individually in a plant,or in combinations of multiple enhanced characteristics.

Improvements in chlorophyll content can be determined by variousmethods. In some aspects, the measurement of chlorophyll contentutilizes non-destructive tissue tests. Non-destructive tissue tests canbe performed easily in the field and provide results much faster thanlaboratory tests. There are a number of chlorophyll content meters thatare used for these tests. The meters determine chlorophyll content byshining a light through a leaf inserted in a slot and measuring theamount of light transmitted.

Chlorophyll meters use different units of measure. For instance, whileMinolta uses “SPAD units”, Force-A uses the Dualex Unit and ADC uses aChlorophyll Content Index. All measure essentially the same thing, andconversion tables are available.

In other aspects, chlorophyll content is measured using chlorophyllfluorescence. In his scientific paper Gitelson (1999) states, “The ratiobetween chlorophyll fluorescence, at 735 nm and the wavelength range 700nm to 710 nm, F735/F700 was found to be linearly proportional to thechlorophyll content (with determination coefficient, r2, more than 0.95)and thus this ratio can be used as a precise indicator of chlorophyllcontent in plant leaves.” See Gitelson, Anatoly A; Buschmann, Claus;Lichtenthaler, Hartmut K (1999). “The Chlorophyll Fluorescence RatioF735/F700 as an Accurate Measure of the Chlorophyll Content in Plants”.Remote Sensing of Environment. 69 (3): 296. The fluorescent ratiochlorophyll content meters use this technique to perform measurements.

Chlorophyll fluorometers are designed to measure variable fluorescenceof photosystem II, or PSII. With most types of plant stress, thisvariable fluorescence can be used to measure the level of plant stress.The most commonly used protocols include: Fv/Fm, a dark-adaptedprotocol, Y(II) or ΔF/Fm′ a light adapted test that is used duringsteady state photosynthesis, and various OJIP, dark adapted protocolsthat follow different schools of thought. Longer fluorescence quenchingprotocols can also be used for plant stress measurement, but because thetime required for a measurement is extremely long, only small plantpopulations can generally be tested. NPQ or non-photochemical quenchingis the most popular of these quenching parameters, but other parametersand other quenching protocols are also used.

Another test protocol based on fluorescence is the OJIP test. Thismethod analyses the increase in fluorescence emitted from dark-adaptedleaves when they are illuminated. The rise in fluorescence during thefirst second of illumination follows a curve with intermediate peaks,called the O, J, I, and P steps. In addition, the K step appears duringspecific types of stress, such as N-deficiency. Research has shown the Kstep is able to measure N-stress.

In some embodiments, a liquid composition can be administered before theseed is planted. In some embodiments, a liquid composition can beadministered at the time the seed is planted. In some embodiments, aliquid composition can be applied by dip treatment of the roots. In someembodiments, a liquid composition can be administered to plants thathave emerged from the ground. In some embodiments, a liquid or driedcomposition can be applied to the soil before, during, or after theplanting of a seed. In some embodiments a liquid or dried compositioncan be applied to the soil before or after a plant emerges from thesoil.

In some embodiments, the volume or mass of the microalgae-basedcomposition applied to a seed, seedling, or plant may not increase ordecrease during the growth cycle of the plant (i.e., the amount of themicroalgae composition applied to the plant will not change as the plantgrows larger). In some embodiments, the volume or mass of themicroalgae-based composition applied to a seed, seedling, or plant canincrease during the growth cycle of the plant (i.e., applied on a massor volume per plant mass basis to provide more of the microalgaecomposition as the plant grows larger). In some embodiments, the volumeor mass of the microalgae-based composition applied to a seed, seedling,or plant can decrease during the growth cycle of the plant (i.e.,applied on a mass or volume per plant mass basis to provide more of themicroalgae composition as the plant grows larger).

In one non-limiting embodiment, the administration of the compositionmay comprise contacting the foliage of the plant with an effectiveamount of the composition. In some embodiments, the liquid compositionmay be sprayed on the foliage by a hand sprayer, a sprayer on anagriculture implement, or a sprinkler. In some embodiments, thecomposition can be applied to the soil.

The foliar treatment formulations usable in accordance with theinvention can be used to treat either directly or after precedingdilution with water. The foliar treatment preparations usable inaccordance with the invention or the dilute preparations thereof canalso be used to dress seed of transgenic plants.

For foliar treatment a variety of applications may be used. In oneembodiment the application method is selected from the group comprisingof spray application, drip-and-drench application, and chemigation. Inone embodiment, spray application is preferred.

The rate of application of the composition at the desired concentrationcan be expressed as a volume per area. In some embodiments, the rate ofapplication of the liquid composition in a foliar application cancomprise a rate in the range of 10-50 gallons/acre. In some embodiments,the rate of application of the liquid composition in a foliarapplication can comprise a rate in the rage of 10-15 gallons/acre. Insome embodiments, the rate of application of the liquid composition in afoliar application can comprise a rate in the range of 15-20gallons/acre. In some embodiments, the rate of application of the liquidcomposition in a foliar application can comprise a rate in the range of20-25 gallons/acre. In some embodiments, the rate of application of theliquid composition in a foliar application can comprise a rate in therange of 25-30 gallons/acre. In some embodiments, the rate ofapplication of the liquid composition in a foliar application cancomprise a rate in the range of 30-35 gallons/acre. In some embodiments,the rate of application of the liquid composition in a foliarapplication can comprise a rate in the range of 35-40 gallons/acre. Insome embodiments, the rate of application of the liquid composition in afoliar application can comprise a rate in the range of 40-45gallons/acre. In some embodiments, the rate of application of the liquidcomposition in a foliar application can comprise a rate in the range of45-50 gallons/acre.

In some embodiments, the rate of application of the liquid compositionin a soil or foliar application can comprise a rate in the range of0.01-10 gallons/acre. In some embodiments, the rate can be 0.12-4%. Insome embodiments, the rate of application of the liquid composition in afoliar application may comprise a rate in the range of 0.01-0.1gallons/acre. In some embodiments, the rate of application of the liquidcomposition in a soil or foliar application may comprise a rate in therange of 0.1-1.0 gallons/acre. In some embodiments, the rate ofapplication of the liquid composition in a foliar application maycomprise a rate in the range of 0.25-2 gallons/acre. In someembodiments, the rate of application of the liquid composition in afoliar application may comprise a rate in the range of 1-2 gallons/acre.In some embodiments, the rate of application of the liquid compositionin a foliar application may comprise a rate in the range of 2-3gallons/acre. In some embodiments, the rate of application of the liquidcomposition in a foliar application may comprise a rate in the range of3-4 gallons/acre. In some embodiments, the rate of application of theliquid composition in a foliar application may comprise a rate in therange of 4-5 gallons/acre. In some embodiments, the rate of applicationof the liquid composition in a foliar application may comprise a rate inthe range of 5-10 gallons/acre.

In some embodiments, the v/v ratio of the composition can be between0.001%-50%. The v/v ratio can be between 0.01-25%. The v/v ratio of thecomposition can be between 0.03-10%.

In another non-limiting embodiment, the administration of thecomposition can include contacting the soil in the immediate vicinity ofthe planted seed with an effective amount of the composition. In someembodiments, the liquid composition can be supplied to the soil byinjection into a low volume irrigation system, such as but not limitedto a drip irrigation system supplying water beneath the soil throughperforated conduits or at the soil level by fluid conduits hanging abovethe ground or protruding from the ground. In some embodiments, theliquid composition can be supplied to the soil by a soil drench methodwherein the liquid composition is poured on the soil.

The composition can be diluted to a lower concentration for an effectiveamount in a soil application by mixing a volume of the composition in avolume of water. The percent solids of microalgae sourced componentsresulting in the diluted composition can be calculated by themultiplying the original concentration in the composition by the ratioof the volume of the composition to the volume of water. Alternatively,the grams of microalgae sourced components in the diluted compositioncan be calculated by the multiplying the original grams of microalgaesourced components per 100 mL by the ratio of the volume of thecomposition to the volume of water.

The rate of application of the composition at the desired concentrationcan be expressed as a volume per area. In some embodiments, the rate ofapplication of the liquid composition in a soil application can includea rate in the range of 50-150 gallons/acre. In some embodiments, therate of application of the liquid composition in a soil application caninclude a rate in the range of 75-125 gallons/acre. In some embodiments,the rate of application of the liquid composition in a soil applicationcan include a rate in the range of 50-75 gallons/acre. In someembodiments, the rate of application of the liquid composition in a soilapplication can include a rate in the range of 75-100 gallons/acre. Insome embodiments, the rate of application of the liquid composition in asoil application can include a rate in the range of 100-125gallons/acre. In some embodiments, the rate of application of the liquidcomposition in a soil application can include a rate in the range of125-150 gallons/acre.

In some embodiments, the rate of application of the liquid compositionin a soil application can include a rate in the range of 10-50gallons/acre. In some embodiments, the rate of application of the liquidcomposition in a soil application can include a rate in the range of10-20 gallons/acre. In some embodiments, the rate of application of theliquid composition in a soil application can include a rate in the rangeof 20-30 gallons/acre. In some embodiments, the rate of application ofthe liquid composition in a soil application can include a rate in therange of 30-40 gallons/acre. In some embodiments, the rate ofapplication of the liquid composition in a soil application can includea rate in the range of 40-50 gallons/acre.

In some embodiments, the rate of application of the liquid compositionin a soil application can include a rate in the range of 0.01-10gallons/acre. In some embodiments, the rate of application of the liquidcomposition in a soil application can include a rate in the range of0.01-0.1 gallons/acre. In some embodiments, the rate of application ofthe liquid composition in a soil application can include a rate in therange of 0.1-1.0 gallons/acre. In some embodiments, the rate ofapplication of the liquid composition in a soil application can includea rate in the range of 1-2 gallons/acre. In some embodiments, the rateof application of the liquid composition in a soil application caninclude a rate in the range of 2-3 gallons/acre. In some embodiments,the rate of application of the liquid composition in a soil applicationcan include a rate in the range of 3-4 gallons/acre. In someembodiments, the rate of application of the liquid composition in a soilapplication can include a rate in the range of 4-5 gallons/acre. In someembodiments, the rate of application of the liquid composition in a soilapplication can include a rate in the range of 5-10 gallons/acre.

In some embodiments, the rate of application of the liquid compositionin a soil application can include a rate in the range of 2-20liters/acre. In some embodiments, the rate of application of the liquidcomposition in a soil application can include a rate in the range of3.7-15 liters/acre. In some embodiments, the rate of application of theliquid composition in a soil application can include a rate in the rangeof 2-5 liters/acre. In some embodiments, the rate of application of theliquid composition in a soil application can include a rate in the rangeof 5-10 liters/acre. In some embodiments, the rate of application of theliquid composition in a soil application can include a rate in the rangeof 10-15 liters/acre. In some embodiments, the rate of application ofthe liquid composition in a soil application can include a rate in therange of 15-20 liters/acre.

Many plants can benefit from the application of liquid compositions thatprovide a bio-stimulatory effect. Non-limiting examples of plantfamilies that can benefit from such compositions include plants from thefollowing: Solanaceae, Fabaceae (Leguminosae), Poaceae, Roasaceae,Vitaceae, Brassicaeae (Cruciferae), Caricaceae, Malvaceae, Sapindaceae,Anacardiaceae, Rutaceae, Moraceae, Convolvulaceae, Lamiaceae,Verbenaceae, Pedaliaceae, Asteraceae (Compositae), Apiaceae(Umbelliferae), Araliaceae, Oleaceae, Ericaceae, Actinidaceae,Cactaceae, Chenopodiaceae, Polygonaceae, Theaceae, Lecythidaceae,Rubiaceae, Papveraceae, Illiciaceae Grossulariaceae, Myrtaceae,Juglandaceae, Bertulaceae, Cucurbitaceae, Asparagaceae (Liliaceae),Alliaceae (Liliceae), Bromeliaceae, Zingieraceae, Muscaceae, Areaceae,Dioscoreaceae, Myristicaceae, Annonaceae, Euphorbiaceae, Lauraceae,Piperaceae, Proteaceae, and Cannabaceae.

The Solanaceae plant family includes a large number of agriculturalcrops, medicinal plants, spices, and ornamentals in its over 2,500species. Taxonomically classified in the Plantae kingdom, Tracheobionta(subkingdom), Spermatophyta (superdivision), Magnoliophyta (division),Manoliopsida (class), Asteridae (subclass), and Solanales (order), theSolanaceae family includes, but is not limited to, potatoes, tomatoes,eggplants, various peppers, tobacco, and petunias. Plants in theSolanaceae can be found on all the continents, excluding Antarctica, andthus have a widespread importance in agriculture across the globe.

The Rosaceae plant family includes flowering plants, herbs, shrubs, andtrees. Taxonomically classified in the Plantae kingdom, Tracheobionta(subkingdom), Spermatophyta (superdivision), Magnoliophyta (division),Magnoliopsida (class), Rosidae (subclass), and Rosales (order), theRosaceae family includes, but is not limited to, almond, apple, apricot,blackberry, cherry, nectarine, peach, plum, raspberry, strawberry, andquince.

The Fabaceae plant family (also known as the Leguminosae) comprises thethird largest plant family with over 18,000 species, including a numberof important agricultural and food plants. Taxonomically classified inthe Plantae kingdom, Tracheobionta (subkingdom), Spermatophyta(superdivision), Magnoliophyta (division), Manoliopsida (class), Rosidae(subclass), and Fabales (order), the Fabaceae family includes, but isnot limited to, soybeans, beans, green beans, peas, chickpeas, alfalfa,peanuts, sweet peas, carob, and liquorice. Plants in the Fabaceae familycan range in size and type, including but not limited to, trees, smallannual herbs, shrubs, and vines, and typically develop legumes. Plantsin the Fabaceae family can be found on all the continents, excludingAntarctica, and thus have a widespread importance in agriculture acrossthe globe. Besides food, plants in the Fabaceae family can be used toproduce natural gums, dyes, and ornamentals.

The Poaceae plant family supplies food, building materials, andfeedstock for fuel processing. Taxonomically classified in the Plantaekingdom, Tracheobionta (subkingdom), Spermatophyta (superdivision),Magnoliophyta (division), Liliopsida (class), Commelinidae (subclass),and Cyperales (order), the Poaceae family includes, but is not limitedto, flowering plants, grasses, and cereal crops such as barely, corn,lemongrass, millet, oat, rye, rice, wheat, sugarcane, and sorghum. Typesof turf grass found in Arizona include, but are not limited to, hybridBermuda grasses (e.g., 328 tifgrn, 419 tifway, tif sport).

The Vitaceae plant family includes flowering plants and vines.Taxonomically classified in the Plantae kingdom, Tracheobionta(subkingdom), Spermatophyta (superdivision), Magnoliophyta (division),Magnoliopsida (class), Rosidae (subclass), and Rhammales (order), theVitaceae family includes, but is not limited to, grapes.

The present invention is further illustrated by the following examplesthat should not be construed as limiting. The contents of allreferences, patents, and published patent applications cited throughoutthis application, as well as the Figures, are incorporated herein byreference in their entirety for all purposes.

EXAMPLES Example 1. Preparation of PHYCOTERRA® ST (Lysed ChlorellaMicroalgae)

Preparation of the PHYCOTERRA® ST (lysed Chlorella microalgae) followedthe general outline provided in WO 2017/218896 A1 (Shinde et al.)entitled “Microalgae-Based Composition, and Methods of its Preparationand Application to Plants” with a few modifications. After harvest ofthe Chlorella microalgae culture, the cells were washed to reduce theviscosity of the resulting product. Previously, production ofPHYCOTERRA® ST (lysed Chlorella microalgae) without a washing step hadresulted in batches with relatively high amounts of viscosity (see FIG.1 ). In a comparison of a batch of harvested Chlorella cells that werelysed without washing versus those lysed with washing, the washed cellsdemonstrated a marked decrease in viscosity (see FIG. 2 ).

Various methods can be used to lyse the Chlorella cells including usinga bead mill, a shear mill, or a combination of these techniques. Inaddition, the volumes of aqueous solution used to wash the cells canvary with the volume to volume (v/v) ratio of solution to harvestedChlorella cells ranging from 1:1 to 20:1, including any sub-rangethereof and v/v ratios of 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1,10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, and 20:1.Washing of the Chlorella cells can be performed before or afterpasteurization. Various stabilizers can be added to the lysed Chlorellacells including potassium sorbate, phosphoric acid, ascorbic acid,sodium benzoate, or any combination thereof. The final productPHYCOTERRA® ST (lysed Chlorella microalgae) has a concentration in therange of 1%-20% solids by weight (e.g., 1%-17.5%, 1%-15%, 1%-12.5%,1%-10%, 1%-7.5%,1%-5% solids by weight or about 1%, about 2%, about 3%,about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%,about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about17%, about 18%, about 19%, or about 20% solids by weight).

Example 2. Migration of Non-Pathogenic Microbes Towards PHYCOTERRA®ST-Treated Seed

A seed treated with PHYCOTERRA® ST (lysed Chlorella microalgae) wasplaced on one side of an agar plate (0.5% agar), and an untreated seedwas placed on the opposite side of the agar plate. A non-pathogenicmicrobial strain, originally isolated from a healthy alfalfa rootcollected from the fields in Arizona, was inoculated in the center ofthe plate, and the plate was incubated at room temperature for 22 hours.At the end of the 22-hour incubation, growth and migration of thenon-pathogenic microbial strain was determined. As shown in FIG. 3 , thenon-pathogenic microbial strain grew and migrated towards the seedtreated with PHYCOTERRA® ST (lysed Chlorella microalgae). Thus,treatment of seed with PHYCOTERRA® ST (lysed Chlorella microalgae) canlead to the active recruitment of beneficial soil microbes in proximityto the planted seed.

Example 3. PHYCOTERRA® ST (Lysed Chlorella Microalgae) IncreasesCulturable Bacterial Populations in Various Soil Types

Soil samples representing two different soil textures determined by theUSDA NRCS Soil Texture Calculator were collected from different regionsin the United States: 1) loam soil from Granger, Iowa; and 2) sand soilfrom Douglas, Georgia. The following treatments were applied to eachtype of soil: conventional PHYCOTERRA® (whole cell Chlorella microalgae)and PHYCOTERRA® ST (lysed Chlorella microalgae). Culturable bacterialpopulations were counted 3 days after treatment by preparing a series of10-fold dilutions of soil samples before applying them to Petri dishescontaining an agar-based medium and counting the resulting colonies.Untreated soil samples were also applied to the Petri dishes and countedfor comparison.

Both treatments increased the culturable bacterial populations in thesoil samples significantly compared to the untreated control soilsamples with PHYCOTERRA® ST (lysed Chlorella microalgae) having an evengreater effect than PHYCOTERRA® (whole cell Chlorella microalgae) (seeFIGS. 4-5 ). Without wishing to be bound by any theory, thisrevitalization of the native microbiome in the soil by PHYCOTERRA®(whole cell Chlorella microalgae) and PHYCOTERRA® ST (lysed Chlorellamicroalgae) can contribute to a healthier growth substrate for crops byhelping to build soil structure and retain water more efficiently.

Example 4. PHYCOTERRA® ST (Lysed Chlorella Microalgae) Increases TotalBacterial Populations as well as Putative Beneficial Bacterial Speciesin Various Soil Types

Soil samples representing two different soil textures determined by theUSDA NRCS Soil Texture Calculator were collected from different regionsin the United States: 1) loam soil from Granger, Iowa; and 2) sand soilfrom Douglas, Georgia. The following treatments were applied to eachtype of soil: conventional PHYCOTERRA® (whole cell Chlorella microalgae)and PHYCOTERRA® ST (lysed Chlorella microalgae). At 3 days after theapplication, approximately 1 gram of soil sample was collected andstored at −80° C. for future DNA extraction.

A bacterial community profiling analysis was performed with the treatedsamples to further investigate the effects of with PHYCOTERRA® (wholecell Chlorella microalgae) or PHYCOTERRA® ST (lysed Chlorellamicroalgae) compared to those obtained from the same soil leftuntreated. Total DNA was extracted from soil samples before subjectingit to PCR to amplify the V3-V4 regions (˜430 bp) of the 16S rRNA gene.The resulting amplicons (i.e., amplified partial 16S rRNA gene) weresequenced using a pair-end 2×300 bp Illumina MiSeq™ platform. The raw16S rRNA gene sequences from isolated microbial DNA obtained with thedifferent soil types were processed using QIIME2 version 2020.11 (Bolyenet al., 2019). Briefly, single-end reads were imported into QIIME2 andprocessed with DEBLUR (Amir et al., 2017) to quality filter, trim reads,correct errors, and remove PCR chimeras to obtain representativeoperational taxonomic unit (OTU) sequences. DEBLUR clustered theresulting sequences at the 100% similarity cutoff and the consensustaxonomy for each OTU was classified using a Naive Bayes classifiertrained on 16S rRNA gene sequences from the SILVA v132 database (Quastet al., 2013).

Three days after the application, both treatments increased the total(including both culturable and non-culturable) bacterial populations inthe soil samples significantly compared to the untreated control soilsamples with PHYCOTERRA® ST (lysed Chlorella microalgae) having an evengreater effect than PHYCOTERRA® (whole cell Chlorella microalgae) (seeFIGS. 6-7 ). In Georgia soil, the treatment with PHYCOTERRA® ST (lysedChlorella microalgae) have a greater impact than PHYCOTERRA® (whole cellChlorella microalgae) in increasing the population of a bacterialspecies belonging to Bacillus, whereas in Iowa soil the treatment withPHYCOTERRA® ST (lysed Chlorella microalgae) significantly increased abacterial species belonging to Pseudomonas. These bacterial species aregenerally known as beneficial soil bacteria with many representativespecies capable of stimulating plant growth. Therefore, it isadvantageous to apply PHYCOTERRA® ST (lysed Chlorella microalgae) andPHYCOTERRA® (whole cell Chlorella microalgae) to stimulate beneficialsoil microbial populations.

Example 5. PHYCOTERRA® ST (Lysed Chlorella Microalgae) Increases CornShoot Biomass

PHYCOTERRA® ST (lysed Chlorella microalgae) was applied as a seedcoating to corn seed at a rate of 4 ounces per hundredweight (oz/cwt).For comparison, PHYCOTERRA® (whole cell Chlorella microalgae) wasapplied at seeding as an in-furrow application at a rate of 1quart/acre. The PHYCOTERRA® ST (lysed Chlorella microalgae) was appliedat a concentration of about 10.5% solids by weight. Untreated controlseed were planted together with the treated corn seed in a greenhouse.Harvest occurred 39 days after seeding when all plants were at the V-9stage of growth.

The average dry shoot weights were determined for the treated anduntreated plants. A statistical analysis of LSMean Dunnet, p<0.1, wasapplied to determine the statistical significance of any differencesobserved. Surprisingly, PHYCOTERRA® ST-treated plants demonstrated astatistically significant 77% increase in dry shoot weight compared tountreated plants whereas PHYCOTERRA®-treated plants experienced a 7%increase in dry shoot weight (see FIG. 8 ).

Example 6. PHYCOTERRA® ST (Lysed Chlorella Microalgae) Increases LettuceShoot Biomass

PHYCOTERRA® ST (lysed Chlorella microalgae) and PHYCOTERRA® (whole cellChlorella microalgae) were applied as drenches to Romaine lettuce(Valley Heart variety) plants at a concentration of 5% (v/v) at seeding.Control Romaine lettuce (Valley Heart variety) plants were also seededand grown without treatment for comparison. All lettuce plants weregrown for 25 days in a greenhouse and afterwards evaluated for shootbiomasses. Surprisingly, PHYCOTERRA® ST-treated plants demonstrated an85% increase in average shoot biomass compared to untreated plantswhereas PHYCOTERRA®-treated plants experienced a 53% increase in averageshoot biomass (see FIG. 9 ).

Example 7. PHYCOTERRA® ST (Lysed Chlorella Microalgae) Increases theShoot Biomasses of a Clover Cover Crop and a Cereal Rye Cover Crop

PHYCOTERRA® ST (lysed Chlorella microalgae) was applied as a seedcoating at 4 oz/cwt to cover crops consisting of clover or cereal rye.Control clover and cereal rye plants were seeded and grown withouttreatment for comparison. All plants were grown for 20 days in agreenhouse and afterwards evaluated for shoot biomasses. PHYCOTERRA®ST-treated clover demonstrated a 36% increase in average shoot biomasscompared to untreated plants, and PHYCOTERRA® ST-treated cereal ryedemonstrated a 14% increase in average shoot biomass compared tountreated plants (see FIGS. 10 and 11 ). A statistical analysis ofLSMean Dunnet, p<0.1, was applied to determine the statisticalsignificance of any differences observed. This analysis indicated thatthe differences observed with the clover cover crop were statisticallysignificant while the differences observed with the cereal rye covercrop had a p-value of 0.1527.

Example 8. PHYCOTERRA® ST (Lysed Chlorella Microalgae) Wheat FieldTrials

Several independent field trials were conducted in various locationsthroughout the state of North Carolina. In one field trial conducted inHertford, N.C., CROPLAN® 9606 Brand Wheat (Variety 112371W) was treatedwith a standard combination of fertilizer and insecticide alone or incombination with PHYCOTERRA® ST (lysed Chlorella microalgae) at anapplication rate of 5 oz/cwt. The soil in this location was acombination of Nimmo loamy fine sand and Arapahoe fine sandy loam. Theresulting wheat seedlings were evaluated for overall growth. Thephotograph in FIG. 12 shows enhanced shoot growth and root growth in theseedlings treated with PHYCOTERRA® ST (lysed Chlorella microalgae)compared to those seedlings receiving only the fungicide andinsecticide.

In field trials conducted in Hassel, N.C.; Kenansville, N.C.; andBurgaw, N.C., final yields were determined with wheat treated with astandard combination of fertilizer and insecticide alone or incombination with PHYCOTERRA® ST (lysed Chlorella microalgae) at anapplication rate of 5 oz/cwt. The soil at each location was a sandy loamsoil. Application of PHYCOTERRA® ST (lysed Chlorella microalgae)consistently resulted in an increased yield averaging +5% over the wheattreated with only the fungicide and insecticide (see FIG. 13 ).

Example 9. PHYCOTERRA® ST (Lysed Chlorella Microalgae) in Combinationwith PHYCOTERRA® (Whole Cell Chlorella Microalgae) Produces aSynergistic Effect in Corn

The advanced plant growth enhancement activity of the algal compositioncombinations is evident from the example below. While each individualalgal composition exhibits efficacy in enhancing plant growth, thecombination has an activity which exceeds a simple addition ofactivities.

PHYCOTERRA® ST (lysed Chlorella microalgae) was applied as a seedcoating to corn seeds at a rate of 4 oz/cwt. PHYCOTERRA® (whole cellChlorella microalgae) was subsequently applied at seeding at a rate of 1quart per acre (qt/ac). Untreated control plants were compared to plantstreated with PHYCOTERRA® ST (lysed Chlorella microalgae) alone,PHYCOTERRA® (whole cell Chlorella microalgae) alone, and a combinationof the two treatments. Harvest occurred 39 days after seeding when allplants were at the V-9 stage of growth in the greenhouse. The averagedry shoot weights were determined for the treated and untreated plants.

A synergistic effect is present when the plant growth enhancementactivity of the algal composition combination exceeds the total of theactivities of the algal compositions when applied individually. Theexpected activity for a given combination of two plant growthenhancement agents can be calculated as follows (cf. Colby, S. R.,“Calculating Synergistic and Antagonistic Responses of HerbicideCombinations,” Weeds, 1967, 15, 20-22):

If

-   -   X is the plant growth enhancement activity when agent A is        applied at an application rate of m gallons/acre (or        liters/hectare),    -   Y is the plant growth enhancement activity when agent B is        applied at an application rate of n gallons/acre (or        liters/hectare),    -   E is the plant growth enhancement activity when the active        compounds A and B are applied at application rates of m and n        gallons/acre (or liters/hectare), respectively,    -   Then

$E = {X + Y - \frac{X \cdot Y}{100}}$

The degree of plant growth enhancement activity compared to untreatedcontrol, expressed in %, is denoted. 0% means plant growth whichcorresponds to that of the untreated control while an activity of 100%means that the plant growth is twice that observed with the untreatedcontrol.

If the actual plant growth enhancement activity exceeds the calculatedvalue, then the activity of the combination is super additive, i.e., asynergistic effect exists. In this case, the efficacy which was actuallyobserved must be greater than the value for the expected efficacy (E)calculated from the above-mentioned formula.

The results shown in Table 1 clearly indicate a synergistic effectresulting from the combination treatment of plants with PHYCOTERRA® ST(lysed Chlorella microalgae) and PHYCOTERRA® (whole cell Chlorellamicroalgae). Photographs of the shoots and roots comparing untreatedplants to plants treated with the combination of the two productsconfirmed a striking increase in both shoot and root growth (see FIGS.14 and 15 ).

TABLE 1 Measurement of average plant growth enhancement. ApplicationActivity in % Algal Composition(s) Rate(s) Found* Calc.** UntreatedControl — 0 — PHYCOTERRA ® 1 qt/ac 7 — PHYCOTERRA ® ST 4 oz/cwt 77 —PHYCOTERRA ® + 1 qt/ac 103 79 PHYCOTERRA ® ST 4 oz/cwt *Found = activityobserved **Calc. = activity calculated using Colby's formula

Example 10. PHYCOTERRA® ST (Lysed Chlorella Microalgae) in Combinationwith PHYCOTERRA® (Whole Cell Chlorella Microalgae) Produces aSynergistic Effect in Potatoes

PHYCOTERRA® ST (lysed Chlorella microalgae) was applied as a seedcoating to seed potatoes at a rate of 8.3 fluid ounces per hundredweight(fl oz/cwt). PHYCOTERRA® (whole cell Chlorella microalgae) wassubsequently applied at a rate of 4.25 qt/acre by sprayer boom over rowridges. The potatoes used were of the Brooke variety, and the trialoccurred in fields containing sandy loam soil having 2.7% organicmatter. Untreated control plants were compared to plants treated withPHYCOTERRA® ST (lysed Chlorella microalgae) alone, PHYCOTERRA® (wholecell Chlorella microalgae) alone, and a combination of the twotreatments.

At harvest, the marketable yield was determined for each group ashundredweight per acre. The marketable yield values for each group werecompared and reported in terms of a percentage relative to the untreatedcontrol group with 0% indicating no increase in marketable yield overthe untreated control group and 100% indicating a marketable yield twicethat of the untreated control group. Analysis of the results with theColby formula indicates a synergistic effect resulting from thecombination treatment of plants with PHYCOTERRA® ST (lysed Chlorellamicroalgae) and PHYCOTERRA® (whole cell Chlorella microalgae) (see Table2).

TABLE 2 Measurement of marketable yield enhancement. ApplicationActivity in % Algal Composition(s) Rate(s) Found* Calc.** UntreatedControl — 0 — PHYCOTERRA ® 4.25 qt/ac 0 — PHYCOTERRA ® ST 8.3 fl oz/cwt5 — PHYCOTERRA ® + 4.25 qt/ac 12 5 PHYCOTERRA ® ST 8.3 fl oz/cwt *Found= activity observed **Calc. = activity calculated using Colby's formula

Example 11. PHYCOTERRA® FX (Lysed Chlorella Microalgae) in Combinationwith PHYCOTERRA® (Whole Cell Chlorella Microalgae) Produces aSynergistic Effect in Lettuce Synergistic Effect on Shoot Growth

PHYCOTERRA® FX (lysed Chlorella microalgae) was applied as a drench tolettuce plants at seeding at a rate of 1 qt/acre. PHYCOTERRA® (wholecell Chlorella microalgae) was also applied as a drench at seeding at arate of 1 quart per acre (qt/ac). Untreated control plants were comparedto plants treated with PHYCOTERRA® FX (lysed Chlorella microalgae)alone, PHYCOTERRA® (whole cell Chlorella microalgae) alone, and acombination of the two treatments.

The average shoot weights of each group of lettuce plants weredetermined several weeks later with treated groups compared and reportedin terms of a percentage relative to the untreated control group with 0%indicating no increase in average shoot weight over the untreatedcontrol group and 100% indicating an average shoot weight twice that ofthe untreated control group. Analysis of the results with the Colbyformula indicates a synergistic effect resulting from the combinationtreatment of plants with PHYCOTERRA® FX (lysed Chlorella microalgae) andPHYCOTERRA® (whole cell Chlorella microalgae) (see Table 3).

TABLE 3 Measurement of shoot growth enhancement. Application Activity in% Algal Composition(s) Rate(s) Found* Calc.** Untreated Control — 0 —PHYCOTERRA ® 1 qt/ac 0 — PHYCOTERRA ® FX 1 qt/ac 9 — PHYCOTERRA ® + 1qt/ac 11 9 PHYCOTERRA ® FX 1 qt/ac *Found = activity observed **Calc. =activity calculated using Colby's formula

Synergistic Effect on Chlorophyll Content

The experiment was repeated as outlined above except that PHYCOTERRA® FX(lysed Chlorella microalgae) was applied as a foliar treatment to thelettuce plants at a rate of 1 qt/acre three days after the PHYCOTERRA®(whole cell Chlorella microalgae) was applied as a drench. Several daysafter both treatments were complete, the average chlorophyll content ofeach group of lettuce plants was determined in SPAD units with treatedgroups compared and reported in terms of a percentage relative to theuntreated control group with 0% indicating no increase in chlorophyllweight over the untreated control group and 100% indicating achlorophyll content twice that of the untreated control group. Analysisof the results with the Colby formula indicates a synergistic effectresulting from the combination treatment of plants with PHYCOTERRA® FX(lysed Chlorella microalgae) and PHYCOTERRA® (whole cell Chlorellamicroalgae) (see Table 4).

TABLE 4 Measurement of chlorophyll content enhancement. ApplicationActivity in % Algal Composition(s) Rate(s) Found* Calc.** UntreatedControl — 0 — PHYCOTERRA ® 1 qt/ac 0 — PHYCOTERRA ® FX 1 qt/ac 0 —PHYCOTERRA ® + 1 qt/ac 3 0 PHYCOTERRA ® FX 1 qt/ac *Found = activityobserved **Calc. = activity calculated using Colby's formula

Unless defined otherwise, all technical and scientific terms herein havethe same meaning as commonly understood by one of ordinary skill in theart to which this invention belongs. Although any methods and materials,similar or equivalent to those described herein, can be used in thepractice or testing of the present invention, the preferred methods andmaterials are described herein. All publications, patents, and patentpublications cited are incorporated by reference herein in theirentirety for all purposes.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth and as follows in the scope ofthe appended claims.

REFERENCES

-   Amir A, McDonald D, Navas-Molina JA, Kopylova E, Morton J T, Zech Xu    Z, Kightley E P, Thompson L R, Hyde E R, Gonzalez A, Knight R. 2017.    Deblur rapidly resolves single-nucleotide community sequence    patterns. mSystems 2:e00191-16.-   Bolyen, E., Rideout, J. R., Dillon, M. R. et al. Reproducible,    interactive, scalable and extensible microbiome data science using    QIIME 2. Nat Biotechnol 37, 852-857 (2019).-   Quast, C., E. Pruesse, P. Yilmaz, J. Gerken, T. Schweer, P.    Yarza, J. Peplies and F. O. Glöckner (2013). “The SILVA ribosomal    RNA gene database project: improved data processing and web-based    tools.” Nucleic Acids Research 41(Database issue): D590-D596.

What is claimed is:
 1. A method comprising: a) applying an effectiveamount of a first liquid composition to a locus surrounding a plant,said first liquid composition comprising a culture of microalgaecomprising whole pasteurized Chlorella cells; and b) applying aneffective amount of a second liquid composition to the locus surroundingthe plant, said second liquid composition comprising a culture ofmicroalgae comprising lysed pasteurized Chlorella cells, whereinapplication of the first liquid composition to the locus surrounding theplant in combination with application of the second liquid compositionto the locus surrounding the plant produces synergistic enhancement ofat least one characteristic of the plant.
 2. The method of claim 1,wherein the first liquid composition and the second liquid compositionare applied simultaneously.
 3. The method of claim 1, wherein the firstliquid composition and the second liquid composition are appliedsequentially.
 4. The method of claim 1, wherein the first liquidcomposition and the second liquid composition are applied as a soildrench or in-furrow treatment.
 5. The method of claim 1, wherein thewhole pasteurized Chlorella cells and/or the lysed pasteurized Chlorellacells are pasteurized at a temperature ranging from 50° C. to 90° C. 6.The method of claim 1, wherein the lysed pasteurized Chlorella cells arelysed with a bead mill, a shear mill, a pulsed electron field (PEF),high pressure homogenization, an enzyme, a chemical solvent, or acombination thereof.
 7. The method of claim 1, wherein the first liquidcomposition and/or the second liquid composition further comprise atleast one culture stabilizer.
 8. The method of claim 7, wherein the atleast one culture stabilizer comprises one or more of potassium sorbate,phosphoric acid, ascorbic acid, sodium benzoate, and citric acid.
 9. Themethod of claim 7, wherein the at least one culture stabilizer is a pHstabilizer.
 10. The method of claim 7, wherein the at least one culturestabilizer prevents growth of contaminants.
 11. The method of claim 1,wherein the at least one characteristic comprises one or more of:accelerated seed germination, accelerated seedling emergence, improvedseedling emergence, improved leaf formation, accelerated leaf formation,improved plant maturation, accelerated plant maturation, increased plantyield, increased plant growth, increased plant quality, increased planthealth, increased fruit yield, increased fruit sweetness, increasedfruit growth, and increased fruit quality
 12. The method of claim 11,wherein the at least one characteristic comprises one or more of: shootgrowth, root growth, marketable yield, and chlorophyll content.